Certain 1-(D-cycloproplyglycinyl)-4-piperidin-4-yl)piperazine compounds as inhibitors of the serine protease factor Xa

ABSTRACT

The compounds of formula (I) 
                         
in which R represents a hydrogen atom or a fluorine atom, or a pharmaceutically acceptable salt thereof are Factor Xa inhibitors useful in the treatment of thrombotic disorders.

This application is the national stage of PCT/US03/07794, filed Mar. 24,2003, and claims the benefit of United States provisional patentapplication Ser. No. 60/368,523, filed

The present invention relates to compounds useful as pharmaceuticals, topharmaceutical compositions comprising the compounds, to a process forpreparing the compounds, to intermediates useful in the preparation ofthe compounds, and to use of the compounds as pharmaceuticals.

Cardiovascular disease continues to present a major worldwide healthproblem, and is a common cause of serious illness and death.

One line of investigation being pursued by researchers in the search fornew treatments for cardiovascular disease is based upon the hypothesisthat an inhibitor of the serine protease, Factor Xa, may be useful as ananticoagulant agent in the treatment of thrombotic disease.

Inhibitors of Factor Xa are known. For example, WO 01/96323 disclosecertain compounds containing an aromatic group, a glycine residue thatbears a cyclic group and a 4-substituted piperazinyl, group. The cyclicgroup may be a cycloalkyl group, such as cyclopentyl or cyclohexyl, buta preference is expressed for compounds in which the cyclic group is aphenyl group.

Surprisingly, it has now been found that by selecting from within thescope of WO 01/96323 a 4-methoxyphenyl or 3-fluoro-4-methoxyphenyl groupas the aromatic group, a cyclopropylglycine group as the glycine residueand a 1-methylpiperidin-4-ylpiperazinyl group as the 4-substitutedpiperazinyl group, compounds may be obtained that are selective FactorXa inhibitors and have particularly advantageous properties.

Accordingly, the present invention provides a compound of formula (I)

in which R represents a hydrogen atom or a fluorine atom, or apharmaceutically acceptable salt thereof.

The compounds of formula (I) have been found to be potent and selectiveinhibitors of the serine protease, Factor Xa, to have good anticoagulantactivity in human plasma, to have good plasma exposure upon oraladministration to mammals, and to possess particularly advantageouspharmacological and toxicological profiles of activity.

The compounds of formula (I) may also be referred to by the chemicalnames1-(4-methoxybenzoyl-D-cyclopropyl-glycinyl)-4-(1-methylpiperidin-4-yl)piperazineand1-(3-fluoro-4-methoxybenzoyl-D-cyclopropyl-glycinyl)-4-(1-methylpiperidin-4-yl)piperazine.

It will be appreciated that the compounds of formula (I) contain acenter of asymmetry that has the (D) configuration. The compounds maytherefore exist and be isolated in a mixture with the corresponding (L)isomer, such as a racemic mixture, or separately. Preferably thecompounds are isolated substantially free of the (L) isomers.

It will also be appreciated that the compounds of formula (I) or theirpharmaceutically acceptable salts may be isolated in the form ofsolvates, and accordingly that any such solvate is included within thescope of the present invention.

Examples of pharmaceutically acceptable salts include hydrochloride,fumarate and maleate acid addition salts.

It will be appreciated that the compounds of formula (I) contain twobasic nitrogen atoms and may therefore form mono and di-acid additionsalts.

One group of compounds of formula (I) is that in which R is a hydrogenatom.

Particular compounds in which R represents a hydrogen atom are:

-   1-(4-methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine    and the hydrochloride, fumarate and maleate acid addition salts    thereof, especially the dihydrochloride, difumarate and dimaleate    acid addition salts.

Crystalline dihydrochloride, difumarate and dimaleate acid additionsalts have been prepared and characterized by differential scanningcalorimetry (DSC):

-   dihydrochloride: decomposes before melting;-   difumarate: 222.8° C. (onset), 225.4° C. (peak); and-   dimaleate: 195.8° C. (onset), 202.0° C. (peak).

Particular mention is made of1-(4-methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)piperazinedifumarate in crystalline form.

Another group of compounds of formula (I) is that in which R is afluorine atom.

Particular compounds in which R represents a fluorine atom are:

-   1-(3-fluoro-4-methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine;    and the hydrochloride acid addition salts.

The compounds of formula (I) and their pharmaceutically acceptable saltsmay be prepared by a process which comprises:

(a) reacting a compound of formula (II)

or a salt thereof (such as the trihydrochloride), with a compound offormula (III)

or a reactive derivative thereof; or

(b) reacting a compound of formula (IV)

or a salt thereof (such as the dihydrobromide), with a compound offormula (V)

or a reactive derivative thereof;

followed, if a pharmaceutically acceptable salt is desired, by forming apharmaceutically acceptable salt.

The reaction between the compound of formula (II) with the compound offormula (III) may conveniently be performed employing reagents andreaction conditions conventionally used for the formation of an amidebond. The reaction is conveniently carried out in the presence of abenzotriazole-based reagent such as 1-hydroxybenzotriazole or1-hydroxy-7-azabenzotriazole and a dehydrating agent such asdicyclohexylcarbodiimide or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, in an inert organicsolvent such as dimethylformamide and/or methylene chloride. Thereaction is conveniently conducted at a temperature of from 0 to 50° C.,preferably at ambient temperature. If a salt of a compound of formula(II) is used, the reaction is conveniently performed in the additionalpresence of a base such as triethylamine. Other suitable reagents andsolvents are known in the art, for example an acid halide, such asp-anisoyl chloride in the presence of a base, such as triethylamine.

The reaction between the compound of formula (IV) with the compound offormula (V) may conveniently be performed employing reagents andreaction conditions conventionally used for the formation of an amidebond, for example as described above for the reaction of a compound offormula (II) with a compound of formula (III).

The compounds of formula (II) may be prepared by reacting a compound offormula (IV) with a compound of formula (VI)

in which R¹ represents an amino protecting group, such ast-butoxycarbonyl (Boc) to afford a compound of formula (VII)

followed by removing the protecting group.

The compound of formula (IV) is known, and is also referred to as1-(1-methylpiperidin-4-yl)piperazine.

The compounds of formula (V) may be prepared by reacting a compound offormula (VIII)

in which R² represents a carboxyl protecting group, for example a(1–6C)alkyl group, such as methyl or ethyl, with a compound of formula(III) to afford a compound of formula (IX)

followed by removing the protecting group.

The compounds of formulae (VI) and (VIII) are known or may be preparedfrom (D)-cyclopropylglycine using conventional methods for theprotection of the carboxy or amino group in an amino acid.(D)-Cyclopropylglycine may conveniently be prepared fromcyclopropanecarboxaldehyde using (R)-(+)-α-methylbenzylamine by themethod described in U.S. Pat. No. 6,090,982, or by using a procedurereferenced therein.

The compounds of formula (III) are well known.

The protection of amino and carboxylic acid groups is described inMcOmie, Protecting Groups in Organic Chemistry, Plenum Press, NY, 1973,and Greene and Wuts, Protecting Groups in Organic Synthesis, 2nd. Ed.,John Wiley & Sons, NY, 1991. Examples of carboxy protecting groupsinclude C₁–C₆ alkyl groups such as methyl, ethyl, t-butyl and t-amyl;aryl(C₁–C₄)alkyl groups such as benzyl, 4-nitro-benzyl, 4-methoxybenzyl,3,4-dimethoxybenzyl, 2,4-dimethoxy-benzyl, 2,4,6-trimethoxybenzyl,2,4,6-trimethylbenzyl, benzhydryl and trityl; silyl groups such astrimethylsilyl and t-butyldimethylsilyl; and allyl groups such as allyland 1-(trimethylsilylmethyl)prop-1-en-3-yl.

Examples of amine protecting groups include acyl groups, such as groupsof formula R³CO in which R³ represents C₁₋₆ alkoxy, phenyl C₁₋₆ alkoxy,or a C₃₋₁₀ cycloalkoxy, wherein a phenyl group may be optionallysubstituted, for example by one or two of halogen, C₁–C₄ alkyl and C₁–C₄alkoxy.

Preferred amino protecting groups include benzyloxycarbonyl (CBz) andt-butoxycarbonyl (Boc).

Certain of the intermediates described herein, for example the compoundsof formulae (II) and (V), are believed to be novel and accordingly areprovided as further aspects of the invention.

The compounds of the invention may be administered by any convenientroute, e.g. into the gastrointestinal tract (e.g. rectally or orally),the nose, lungs, musculature or vasculature or transdermally. Thecompounds may be administered in any convenient administrative form,e.g. tablets, powders, capsules, solutions, dispersions, suspensions,syrups, sprays, suppositories, gels, emulsions, patches etc. Suchcompositions may contain components conventional in pharmaceuticalpreparations, e.g. diluents, carriers, pH modifiers, sweeteners, bulkingagents, and further active agents. If parenteral administration isdesired, the compositions will be sterile and in a solution orsuspension form suitable for injection or infusion. Such compositionsform a further aspect of the invention.

Viewed from this aspect the invention provides a pharmaceuticalcomposition, which comprises the compound of formula (I) or apharmaceutically acceptable salt thereof, together with apharmaceutically acceptable diluent or carrier.

According to another aspect, the present invention provides the compoundof formula (I) or a pharmaceutically acceptable salt thereof, for use intherapy.

According to another aspect, the present invention provides the use ofthe compound of formula (I) or a pharmaceutically acceptable saltthereof, for the manufacture of a medicament for the treatment of athrombotic disorder.

According to another aspect, the present invention provides a method oftreating a thrombotic disorder in a subject requiring treatment, whichcomprises administering an effective amount of a compound of formula (I)or a pharmaceutically acceptable salt thereof.

The subject may be a human or a non-human animal, such as a non-humanmammal, for example a cat, dog, horse, cow or sheep.

The thrombotic disorder may be, for example, venous thrombosis,pulmonary embolism, arterial thrombosis, myocardial ischaemia,myocardial infarction or cerebral thrombosis. The compounds may also beused in accordance with the method of the invention in the treatment ofacute vessel closure associated with thrombolytic therapy andrestenosis, for example after transluminal coronary angioplasty orbypass grafting of the coronary or peripheral arteries, and in themaintenance of vascular access patency in long term hemodialysispatients.

The dosage of the compound of formula (I) will depend upon the natureand severity of the condition being treated, the administration routeand the size and species of the subject. In general, quantities in therange of from 0.01 to 100 μM/kg bodyweight will be administered.

As used herein, the term “treatment” includes prophylactic use. The term“effective amount” refers to the amount of the compound of formula (I)that is effective to reduce or inhibit the development of the symptomsof the thrombotic disorder being treated.

The compound according to the invention may be administered alone or incombination with an anticoagulant having a different mode of action orwith a thrombolytic agent.

The following Examples illustrate the invention.

Abbreviations used follow IUPAC-IUB nomencalture. The followingabbreviations are used throughout: Boc (tertiary-butyloxycarbonyl),Calcd (calculated), DMSO (dimethyl sulfoxide, perdeuterated if for NMR),EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride),ES-MS (electron spray ioniazation mass spectrum), HOBt(1-hydroxy-benzotriazole), HPLC (high-performance liquid chromatographywith t_(r) as retention time), MeOH (methanol), NMR (nuclear magneticresonance), TFA (trifluoroacetic acid).

EXAMPLE 11-(4-Methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazine.A. D-Cyclopropylglycine

The amino acid is obtained from cyclopropane-carboxaldehyde,conveniently by using (R)-(+)-α-methyl-benzylamine and the procedure ofU.S. Pat. No. 6,090,982, or by using a procedure referenced therein.

B. Boc-D-cyclopropylglycine

A solution of D-cyclopropylglycine (46.1 g, 0.4 mol) in a mixture ofdioxane (600 mL), water (300 mL) and 1 N NaOH (480 mL, 0.48 mol) isstirred cooled to 0–5° C. in an ice bath. Di-tert-butyl dicarbonate (105g, 0.48 mol) is added slowly, and stirring is continued at roomtemperature for 0.5 h. The solution is concentrated in vacuo to about500 mL, cooled in an ice-water bath, covered with a layer of ethylacetate (500 mL) and acidified with a dilute aqueous solution of KHSO₄to pH 2–3. The aqueous phase is extracted with ethyl acetate (500 mL)and the extraction repeated until no product remains. The ethyl acetateextracts are pooled, washed with water (0.5 L), brine (0.5 L), driedover Na₂SO₄, filtered and concentrated to give a white solid (78 g,90.6%). [α]_(D) ²⁰=−31° (c=1.02, MeOH).

¹H NMR (CDCl₃) δ 5.9 (sb, 1H), 5.09 (sb, 1H), 3.75 (m, 1H), 1.42 (s,9H), 1.10 (d, 1H), 0.4–0.7 (m, 4H).

C. 1-(Boc-D-cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine

Boc-D-cyclopropylglycine (216 g, 1.0 mol) and1-(1-methylpiperidin-4-yl)piperazine (192 g, 1.05 mol) are slurried inanhydrous CH₂Cl₂ (3.2 L) under N₂. The mixture is then cooled to 0–5° C.in an ice bath. To this mixture, 1-hydroxybenzotriazole (HOBt)monohydrate (149 g, 1.1 mol) and diisopropylethylamine (136 g, 1.05 mol)are added, followed by slow addition of1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI)(211 g, 1.1 mol) maintaining temp at 0–5° C. for 1 h. The reactionmixture is allowed to warm to room temperature overnight. The reactionis then quenched with the addition of saturated (satd) aqueous NaHCO₃ (3L) and extracted with methylene chloride (2 L). The layers areseparated. The organic layer is washed again with satd NaHCO₃ (3 L),brine (2 L), dried over MgSO₄, filtered and concentrated to give thecrude product as a viscous oil (415 g, 109%) which is used directly.

¹H NMR (DMSO-d₆) δ 6.91 (d, 1H), 3.97 (t, 1H), 3.41 (sb, 4H), 2.74 (d,2H), 2.40 (sb, 4H), 2.10 (m, 1H), 2.09 (s, 3H), 1.79 (t, 2H), 1.65 (d,2H), 1.40 (m, 2H), 1.36 (s, 9H), 1.05 (m, 1H), 0.93 (d, 1H), 0.40 (m,2H), 0.26 (m, 1H).

D. 1-(D-Cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)-piperazinetrihydrochloride

1-(Boc-D-cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)piperazine(crude, 415 g, 1.0 mol) is dissolved in anhydrous methanol (1.5 L). Tothis solution is added HCl-MeOH (380 g/1.5 L, 10.4 mol) solution at 0°C. The reaction mixture is stirred and slowly warmed to room temperaturefor 2 h. Ethyl acetate (2 L) is then added with stirring. Stirring iscontinued for 1 h at 0–5° C., and product precipitates out as a whitepowder which is filtered and dried under vacuum at 45° C. to give thetitle compound as a white solid (357 g, 91.7%).

¹H NMR (DMSO-d₆+D₂O) δ 8.4 (bs, 1H), 4.5 (bs, 2H), 3.0 (t, 4H), 2.78 (m,1H), 2.74 (s, 3H), 2.35 (m, 2H), 2.20 (m, 1H), 2.08 (m, 2H), 1.06 (bd,1H), 0.60 (m, 4H).

E.1-(4-Methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazine

1-(D-Cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)-piperazinetrihydrochloride (300 g, 0.77 mol) is slurried in anhydrous CH₂Cl₂ (3 L)under N₂. The mixture is then cooled to 0–5° C. in an ice bath.Triethylamine (450 mL, 3.23 mol) is added slowly while maintaining thetemperature at 0–5° C., followed by slow addition of p-anisoyl chloride(142 g, 0.83 mol), again maintaining the temperature at 0–5° C. Thereaction mixture is allowed to warm to room temperature for 2 h. Thereaction is then quenched with the addition of satd NaHCO₃ (1 L), andthe layers are separated. The aqueous layer is then extracted withCH₂Cl₂ (2 L). The organic layers are combined, washed with brine (1 L),dried over MgSO₄, filtered and concentrated to give the title compound(326 g, 102%).

¹H NMR (DMSO-d₆) δ 8.55 (d, J=7.7 Hz, 1H), 7.88 (d, 2H), 6.96 (d, 2H),4.39 (t, 1H), 3.79 (s, 3H), 3.45 (s, 4H), 2.74 (d, 2H), 2.40 (m, 4H),2.09 (s, 3H), 2.08 (m, 1H), 1.79 (dt, 2H), 1.62 (d, 2H), 1.38 (m, 2H),1.26 (m, 1H), 0.43 (m, 2H), 0.35 (m, 2H).

EXAMPLE 1A1-(4-Methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methyl-piperidin-4-yl)piperazineDifumarate

To a solution of1-(4-methoxybenzoyl-D-cyclopropyl-glycinyl)-4-(1-methylpiperidin-4-yl)piperazine(315 g, 0.76 mol) in 95% ethanol (4.2 L) warmed to 65° C. is added asolution of fumaric acid (177 g, 1.52 mol) in hot ethanol (at 65° C.,2.8 L). The final clear solution is stirred at 65° C. and slowly cooleddown to room temperature (over two hours) and then to 0–5° C. The whitecrystals are collected by filtration, washed with 95% ethanol (1 L), anddried under vacuum at 45° C. to provide the title salt (448 g, 91.2%),mp=205–207° C.

¹H NMR (DMSO-d₆) δ 11.35 (s, 1H), 8.58 (d, 1H), 7.86 (d, 2H), 6.96 (d,2H), 6.55 (s, 4H), 4.39 (t, 1H), 3.79 (s, 3H), 3.45 (s, 4H), 3.20 (d,2H), 2.62 (t, 2H), 2.55 (s, 3H), 2.42 (m, 6H), 1.80 (d, 2H), 1.62 (d,2H), 1.28 (m, 1H), 0.43 (m, 2H), 0.35 (m, 2H).

[α]_(D) ²⁰=−37.7° (c=0.836, H₂O).

EXAMPLE 1b 1-(4-Methoxybenzoyl-D-cyclopropylglcinyl)-4-(1-methyl-piperidin-4-yl)piperazine Hydrochloride.

1-(4-Methoxybenzoyl-D-cyclopropylglcinyl)-4-(1-methyl-piperidin-4-yl)piperazine (3.04 g, 6.73 mmol) is dissolved inapproximately 0.2 M HCl (37 mL) and lyophilized to give 2.98 g(quantitative) of the title compound.

Partial ¹H NMR Spectrum (DMSO-d₆) δ 10.63 (br, 1H), 8.59 (br, 1H), 7.88(d, 2H), 6.98 (d, 2H), 4.36 (br t, 1H), 3.82 (s, 3H), 2.70 (s, 3H), 1.29(m, 1H), 0.48 (m, 2H), 0.36 (m, 2H).

ES-MS, m/z 415.5 (M+1)⁺.

Analysis for C₂₃H₃₄N₄O₃.1.25 HCl.1.0H₂O:

Calcd: C, 57.78; H, 7.85; N, 11.72; Cl, 9.30. Found: C, 57.79; H, 7.93;N, 11.74; Cl, 9.64.

Analytical HPLC (Xterra RP18, 4.6×150 cm,

-   10% acetonitrile/water (0.1% TFA) through-   50% acetonitrile/water (0.1% TFA) over 40 min), 1 mL/min: 99%,    t_(r)=10.66 min

EXAMPLE 21-[(3-Fluoro-4-methoxybenzoyl)-D-cyclopropylglcinyl]-4-(1-methylpiperidin-4-yl)piperazine

To a stirring suspension of1-D-cyclopropylglcinyl-4-(1-methylpiperidin-4-yl)piperazinetrihydrochloride (1.5 g, 3.85 mmol) in dichloromethane (30 mL) is addedtriethylamine (1.36 g, 13.5 mmol) followed by 3-fluoro-4-methoxybenzoicacid (0.622 g, 3.66 mmol), HOBt (0.573 g, 4.24 mmol) and EDCI (0.813 g,4.24 mmol). After stirring overnight, the mixture is partitioned betweendichloromethane and saturated aqueous sodium bicarbonate. The organicphase is then washed again with saturated aqueous sodium bicarbonate,followed by brine, then dried with MgSO₄, filtered and concentrated invacuo. The residue is then dissolved in dichloromethane andchromatographed over silica gel, eluting with a gradient of 0–12% 2 Nammonia/methanol in dichloromethane. The pure product containingfractions are combined and concentrated in vacuo to give 1.03 g (61%) ofthe title compound.

ES-MS, m/z 433.5 (M+1)⁺.

Analytical HPLC (Vydac C18, 4.6×150 cm,

-   10% acetonitrile/water (0.1% TFA) through-   50% acetonitrile/water (0.1% TFA) over 40 min), 1 mL/min: 95%,    t_(r)=13.16 min

EXAMPLE 2A1-[(3-Fluoro-4-methoxybenzoyl)-D-cyclopropylglcinyl]-4-(1-methylpiperidin-4-yl)piperazineHydrochloride

1-[(3-Fluoro-4-methoxybenzoyl)-D-cyclopropylglcinyl]-4-(1-methylpiperidin-4-yl)piperazine(1 g, 2.31 mmol) is dissolved in approximately 0.2 M HCl (12.7 mL) andlyophilized to give 1.03 g (quantitative) of the title compound.

Partial ¹H NMR Spectrum (DMSO-d₆) δ 10.36 (br, 1H), 8.73 (d, 1H),7.75–7.83 (m, 2H), 7.25 (t, 1H), 4.36 (t, 1H), 3.90 (s, 3H), 2.70 (s,3H), 1.28 (m, 1H), 0.48 (m, 2H), 0.36 (m, 2H).

ES-MS, m/z 433.3 (M+1)⁺.

Analysis For C₂₃H₃₃FN₄O₃.1.6 HCl.0.5H₂O:

Calcd: C, 55.26; H, 7.18; N, 11.21; F, 3.80; Cl, 11.35; Found: C, 55.31;H, 7.24; N, 11.38; F, 3.64; Cl, 11.36.

Analytical HPLC (Xterra RP18, 4.6×150 cm,

-   10% acetonitrile/water (0.1% TFA) through-   50% acetonitrile/water (0.1% TFA) over 40 min), 1 mL/min: 98%,    t_(r)=8.067 min.    Enzyme Inhibition Assays:

The ability of a test compound to inhibit factor Xa may be evaluated inone or more of the following Enzyme Inhibition assays, or in otherstandard assays known to those skilled in the art.

Enzyme Inhibition Assay

Human factor Xa and human thrombin are purchased from Enzyme ResearchLaboratories (South Bend, Ind., USA). Other proteases are from othercommercial sources. Chromogenic para-nitroanilide peptide proteasesubstrates are purchased from Midwest Biotech (Fishers, Ind., USA).

The binding affinities for human factor Xa are were measured as apparentassociation constants (Kass) derived from protease inhibition kineticsas described previously.^(a,b,c,d) The apparent Kass values are obtainedusing automated (BioMek-1000) dilutions of inhibitors (Kassdeterminations are performed in triplicate at each of four-eightinhibitor concentrations) into 96-well plates and chromogenic substratehydrolysis rates determined at 405 nm using a Thermomax plate readerfrom Molecular Devices (San Francisco). For factor Xa inhibition, theassay protocol is: 50 μL buffer (0.06 M tris, 0.3 M NaCl, pH 7.4); 25 μLinhibitor test solution (in MeOH); 25 μL human factor Xa (32 nM in 0.03M tris, 0.15 M NaCl, 1 mg/mL HSA); finally, 150 μL BzIleGluGlyArgpNA(0.3 mM in water) added within 2 min to start hydrolysis. Final [factorXa] is 3.2 nM. [Free Xa] and [bound Xa] are determined from linearstandard curves on the same plate by use of SoftmaxPro software for eachinhibitor concentration and apparent Kass calculated for each inhibitorconcentration which produced hydrolysis inhibition between 20% and 80%of the control (3.2 nM factor Xa): apparentKass=[E:I]/[E_(f)][I_(f)]=[E_(b)]/[E_(f)][I^(o)−I_(b)]. The apparentKass values so obtained are approximately the inverse of the Ki for therespective inhibitors [1/appKass=app Ki]. The variability of meanapparent Kass values determined at the single substrate concentration is+/−15%. The assay system Km was measured as 0.347 +/−0.031 mM [n=4]; andVmax was 13.11 +/−0.76 μM/min.

Kass values are determined with thrombin and other proteases using thesame protocol with the following enzyme and substrate concentrations:

-   thrombin, 5.9 nM with 0.2 mM BzPheValArgpNA;-   factor XIa, 1.2 nM with 0.4 mM pyroGluProArgpNA;-   factor XIIa, 10 nM with 0.2 mM HDProPheArgpNA;-   plasmin, 3.4 nM with 0.5 mM HDValLeuLyspNA;-   nt-PA, 1.2 nM with 0.8 mM HDIleProArgpNA;-   urokinase, 0.4 nM with 0.4 mM pyroGluGlyArgpNA;-   aPC, 3 nM with 0.174 mM pyroGluProArgpNA;-   plasma kallikrein, 1.9 nM with D-ProPheArgpNA; and-   bovine trypsin, 1.4 nM with 0.18 mM BzPheValArgpNA.    Citations    (a) Sall D J, J A Bastian, S L Briggs, J A Buben, N Y Chirgadze, D K    Clawson, M L Denny, D D Giera, D S Gifford-Moore, R W Harper, K L    Hauser, V J Klimkowski, T J Kohn, H-S Lin, J R McCowan, A D    Palkowitz, G F Smith, M E Richett, K Takeuchi, K J Thrasher, J M    Tinsley, B G Utterback, S-C B Yan, M Zhang. Dibasic    Benzo[b]thiophenes Derivatives as a Novel Class of Active Site    Directed Thrombin Inhibitors. 1. Determination of the Serine    Protease Selectivity, Structure-Activity Relationships and Binding    Orientation. J Med Chem 40 3489–3493 (1997).    (b) Smith G F, T J Craft, D S Gifford-Moore, W J Coffman, K D Kurz,    E Roberts, R T Shuman, G E Sandusky, N D Jones, N Chirgadze, and C V    Jackson. A Family of Arginal Thrombin Inhibitors Related to    Efegatran. Sem. Thrombos. Hemost. 22, 173–183 (1996).

(c) Smith G F, D S Gifford-Moore, T J Craft, N Chirgadze, K JRuterbories, T D Lindstrom, J H Satterwhite. Efegatran: A NewCardiovascular Anticoagulant. In New Anticoagulants for theCardiovascular Patient. Ed. R Pifarre. Hanley & Belfus, Inc.,Philadelphia (1997) pp 265–300.

(d) Sall D J, D L Bailey, J A Bastian, N Y Chirgadze, A C Clemens-Smith,M L Denney, M J Fisher, D D Geira, D S Gifford-Moore, R W Harper, L MJohnson, V J Klimkowski, T J Kohn, H S Lin, J R McCowan, A D Palkowitz,M E Richett, G F Smith, D W Snyder, K Takeuchi, J E Toth, M Zang.Diamino Benzo[b]thiophene Derivatives as a Novel Class of Active SiteDirected Thrombin Inhibitors: 5. Potency, Efficacy and PharmacokineticProperties of Modified C-3 Side Chain Derivatives. J. Med. Chem., 43,649–663 (2000).

The compounds of formula (I) exemplified herein have been found toexhibit a Kass of about 14×10⁶ to 35×10⁶ L/mole in the enzyme inhibitionassay.

The ability of a test compound to elongate Partial Thromboplastin Time(Prothrombin Time) may be evaluated in the following test protocols.

Partial Thromboplastin Time (Prothrombin) Test Protocol

Venous blood is collected into 3.2% (0.109 M) trisodium citratevacutainer tubes at 1 volume of anticoagulant to nine volumes of blood.The blood cells are separated by centrifugation at 700 g for ten minutesto yield plasma, which is frozen at 70° C. until required.

To perform the test, 100 μL of plasma are pipetted into in a glass testtube, 1 μL of test compound in DMSO is added, and allowed to warm to 37°over two minutes. 100 μL of warm (37°) Manchester (tissue thromboplasin)reagent (Helena Biosciences, UK) is added, allowed to equilibrate fortwo minutes. 100 μL of warm (37°) 25 mM calcium chloride solution isadded to initiate clotting. The test tube is tilted three times througha 90° angle every five seconds to mix the reagents and the time to clotformation recorded. Data from a series of observations and test compoundconcentrations are analysed by a SAS statistical analysis program and aCT2 (Concentration required to double clotting time) for each compoundis generated.

The compounds of the invention have been found to significantly elongatethe partial thromboplastin time (Prothrombin time).

Alternative Prothrombin Time and APTT Protocols

Coagulation Determinations: Prothrombin Times and APTT values aredetermined in HUMAN PLASMA with a STA instrument (Stago). BioPT is aspecial non-plasma clotting assay triggered with human tissue factor(Innovin). Possible binding to albumen or to lipid are assessed bycomparing the BioPT effects in the presence/absence of 30 mg/mL humanalbumen (HSA) and 1 mg/mL phosphatidyl choline (PC). Inhibitors aredelivered in 50% aqueous methanol vehicle.APTT Assay

-   75 μL plasma Citrol Baxter-Dade Citrated Normal-   Human Plasma-   25 μL test solution-   75 μL Actin Baxter-Dade Activated Cephaloplastin incubate 2 min min.    @ 37° C.-   75 μl CaCl₂ (0.02 M)    PT Assay-   75 μL plasma-   25 μL test solution-   75 μL saline incubate 1 min. @ 37° C.-   75 μL Innovin Baxter-Dade Recombinant Human Tissue Factor

Further advantageous properties of compounds of formula (I) may bedemonstrated by measuring their pharmacodynamic (PD) and pharmacokinetic(PK) properties in laboratory animal species such as rats and dogsfollowing oral dosing in the fasted and in the fed state.

1. A compound of formula (I)

in which R represents a hydrogen atom or a fluorine atom, or apharmaceutically acceptable salt thereof.
 2. A compound as claimed inclaim 1, in which R represents a hydrogen atom.
 3. A compound as claimedin claim 2, which is selected from:1-(4-methoxybenzoyl-D-cyclopropylglycinyl)-4-(1-methylpiperidin-4-yl)piperazineand the hydrochloride, fumarate and maleate acid addition salts thereof.4. A compound as claimed in claim 3, which is selected from thedihydrochloride, difumarate and dimaleate acid addition salts incrystalline form.
 5. A compound as claimed in claim 4, which is1-(4-methoxybenzoyl-D-cyclo-propylglycinyl)-4-(1-methylpiperidin-4-yl)piperazinedifumarate in crystalline form.
 6. A compound as claimed in claim 1, inwhich R represents a fluorine atom.
 7. A compound as claimed in claim 6,which is selected from:1-(3-fluoro-4-methoxybenzoyl-D-cyclopropylglycinyl)-4(1-methylpiperidin-4-yl)piperazine;and the hydrochloride acid addition salts thereof.
 8. A pharmaceuticalcomposition, which comprises a compound as claimed in any one of claims1 to 7, together with a pharmaceutically acceptable diluent or carrier.9. A process for preparing a compound as claimed in claim 1, whichcomprises: (a) reacting a compound of formula (II)

a salt thereof, with a compound of formula (III)

or a reactive derivative thereof; or (b) reacting a compound of formula(IV)

or a salt thereof, with a compound of formula (V)

or a reactive derivative thereof; followed, if a pharmaceuticallyacceptable salt is desired, by forming a pharmaceutically acceptablesalt.
 10. A compound of formula (II)

or a salt thereof.
 11. A compound of formula (V)

in which R represents a hydrogen atom or a fluorine atom.
 12. A methodof treating a thrombotic disorder selected from venous thrombosis,pulmonary embolism, arterial thrombosis, myocardial ischaemia,myocardial infarction and cerebral thrombosis, in a subject requiringtreatment, which comprises administering an effective amount of acompound as claimed in claim 1.